Proton transport across the mitochondrial inner membrane provides the protonmotive force for ATP synthesis, but the importance of the transport of other ions (e.g. K+, Na+, Ca2+, and anions) in the regulation of bioenergetics has become increasingly evident in recent years. Over the past several years, our focus has been on three main areas, i) characterizing the K+ uptake pathways involved in protection against ischemic damage, ii) understanding how Na+ and Ca2+ dynamics impact energy supply and demand balance, and iii) characterizing how inner membrane oxidant-sensitive energy dissipating channels are activated by stress. While much has been learned using pharmacological tools and manipulation of ion gradients in isolated mitochondria, cells, and intact hearts, a major limitation has been the lack of molecular information about the proteins mediating ion transport in the inner membrane. Based on an exhaustive protein purification and fractionation strategy, and the team approach undertaken during the prior funding period designed to fully characterize the mitochondrial proteome and its modifications during ischemia- reperfusion, we have been successful in identifying a number of novel, high confidence candidates that we believe underlie important K+, Na+ and Ca2+ transport pathways in the mitochondrial inner membrane. Intriguingly, we have also identified novel mitochondrial anion transporters that could prove to be involved in the regulation of mitochondrial function. This project will combine molecular techniques for manipulating the expression levels of mitochondrial proteins identified by mass spectrometry with functional assays in isolated mitochondria and cells to correlate a particular ion transport pathway with its corresponding protein. Based on evidence already obtained by mass spectrometry, we believe we are on track to unequivocally resolve the molecular entities comprising the pore forming subunits of the mitochondrial ATP-sensitive (mitoKATP) and calcium-activated (mitoKCa) potassium channels, and are currently pursuing strong candidates that could mediate mitochondrial sodium calcium exchange (mNCE) and monovalent cation-hydrogen exchange (KHE or NHE). We will also investigate the possible functional role of identified, but uncharacterized, anion transporters that may be involved in mitochondrial volume regulation and the response to oxidative stress. The ultimate goal of the project is to overcome a significant roadblock to progress in the area of mitochondrial biology - the molecular identification of ion transport proteins that are critical to normal function and to the pathophysiology of ischemia-reperfusion. PUBLIC HEALTH RELEVANCE: Cardiac arrhythmias and tissue damage during ischemia and reperfusion have been linked to an impaired ability of mitochondria to maintain energy production for muscle contraction and cellular ion homeostasis. Disruption of the normal transport of ions across the mitochondrial inner membrane is the initiating factor in cell death, yet the proteins responsible for this process have not been identified. This project will identify key ion transport proteins involved in the regulation of mitochondrial energetics and examine their role in the susceptibility to ischemia- reperfusion injury.